Chemical analysis of liquids, such as water, milk and biological fluids is often desirable or necessary for health maintenance and diagnostic treatment. Various compositions and elements to facilitate such analyses are known. Such compositions and elements generally include a reagent composition for determining a substance under analysis, termed an analyte herein. The analyte can be a biological organism or a nonliving chemical substance. This reagent composition, upon interaction with the analyte, provides a detectable change (e.g. dye formation).
Recently, much work has been directed to developing compositions and elements which are useful for rapid and highly quantitative diagnostic or clinical analysis of biological fluids such as whole blood, serum, plasma, urine and the like.
For the rapid and effective diagnosis and treatment of infectious diseases, it is desirable to be able to detect the bacteria causing the disease as rapidly as possible. Infections of the urinary tract are among the most common bacterial diseases, second in frequency only to infections of the respiratory tract. In fact, in many hospitals, urinary tract infections are the most common form of nosocomial infections, often following the use of catheters and various surgical procedures. Most urinary tract infections (UTI) result from ascending infection by microorganisms introduced through the urethra and vary in severity from an unsuspected infection to a condition of severe systemic disease. Such infections are usually associated with bacterial counts of 100,000 (10.sup.5) or more organisms per ml of urine, a condition referred to as significant bacteriuria. Under normal conditions, urine is sterile, although contamination from the external genitalia may contribute up to 1,000 (10.sup.3) organisms per ml in properly collected and transported specimens.
Significant bacteriuria may be present in a number of pathological conditions involving microbial invasion of any of the tissues of the urinary tract, or may result from simple bacterial multiplication in the urine without tissue invasion. The infection may involve a single site such as the urethra, prostate, bladder, or kidney, although frequently it involves more than one site. Infection restricted to the urine may present itself as asymptomatic bacteriuria, i.e., a condition which manifests no overt signs or symptoms of infection. Early treatment of this condition can prevent the development of more serious conditions, e.g., pyelonephritis (inflammation of the kidney and the renal pelvis). The rapid detection of bacteria by a reliable method would therefore facilitate an early and specific diagnosis.
Further, in order to insure that a prescribed antibiotic is in fact effective in treating an infection, repeated tests during therapy are required. The need for simple, rapid bacteriuria tests is thus clear. Moreover, in view of the frequent unsuspected asymptomatic occurrences of UTI among children, pregnant women, diabetics and geriatric populations, diagnosis of which may require collection and testing of several specimens, bacteriuria tests must be sufficiently simple and economical to permit routine performance. Again, this illustrates the need for a rapid and inexpensive bacteriuria detection method.
Known commercial methods for relatively rapid detection of bacteria or other living cells have a number of serious drawbacks. It was during attempts to find solutions to these problems that our colleagues unexpectedly discovered that certain reducible compounds would provide detectable dyes to enable rapid detection of bacteria or other analytes at pH 9 or less. This discovery is the subject of U.S. Ser. No. 699,386 of Belly et al, noted above. As described in that patent application, determination of certain analytes, notably living cells (such as bacteria, yeast, etc.), is best accomplished using a reducible compound which releases a shiftable detectable species to provide a detectable species in the presence of an electron transfer agent (ETA). The electron transfer agent is first reduced by the living cell. The reduced ETA then reduces the reducible compound whereupon the shiftable detectable species is released. Phenazine methosulfate (PMS) is the preferred ETA used by Belly et al in the practice of their invention.
While the Belly et al invention represents a significant advance in the art, PMS and structurally related ETAs are unstable in aqueous solutions. This instability leads to premature release of the shiftable detectable species. This undesirable release is evidenced by high background levels which must be subtracted from assay results to accurately determine the analyte. The background may significantly affect the results when low level analytes (e.g. low levels of bacteria) are being measured.
Other known ETAs which have been tried in biological studies, e.g. pyrocyanine and menadione, are generally ineffective in promoting release of a shiftable detectable species.
Hence, there is a need in the art for a rapid and highly quantitative assay for analytes in aqueous liquids that is not subject to high background levels.